The main objectives of the proposed research are (a) design and study of new degenerate four-wave mixing (D4WM) laser spectroscopic methods in high-resolution measurement of stable isotopes important for biotracer studies, (b) careful comparison of these methods with currently available methods, and (c) the demonstration of the effectiveness and the usefulness of these methods in biotracer studies using real biomedical samples. Although many isotopes have been used in studying mineral absorption and metabolism in human subjects, there is no simple, routine analytical method for measurement of stable isotope enrichment. Currently available methods such as mass spectrometry or ICP-MS have limitations including slow sample throughput, high cost, isobaric or matrix interferences, and time-consuming sample purification or modification (chelation) steps. Safe and reliable biotracer studies can be performed by measuring stable non-radioactive isotopes at trace-concentration levels using D4WM. Laser D4WM offers excellent sub-Doppler (and Lorentzian minimized) spectral resolution, and hence, it can resolve not only isotopes, but also hyperfine lines of individual isotopes. A hyperfine structure, the atomic spectroscopic fingerprint, is unique and no two structures are identical, and hence, it offers unambiguous measurements. Hence, spectral and chemical interference problems are negligible in these high-resolution studies, even when a mixture of many elements is present in the sample. Isotope ratios can be measured by simply comparing the peak intensities in many well-resolved and spread-out hyperfine structures. when hyperfine structures are convoluted, rich isotope information can still be extracted by using spectral deconvolution schemes based on theoretical D4WM calculations. Both stable and radioactive isotopes can be measured simultaneously, since all types of isotopes present are measured in a hyperfine structure. Some of the advantages over conventional methods include: excellent sensitivity, selectivity, reproducibility, relatively inexpensive instrumentation, simple and safe operation, and fast sample throughput. The effectiveness and the accuracy of these new laser methods will be investigated for some of the most important elements in biotracer isotope studies, such as Ca, Pb, Mg, Cu, Ni, Ag, Fe, Cd, Zn, Pd, Re, Os, Li, Ba, Al, and Rb, using different atomizers including discharge, graphite furnace and flame. Careful and detailed studies on the reliability and the usefulness of these new laser-based methods will be performed for many real biosamples while comparing them to currently available methods for biotracer studies. The PI will prove, compare and demonstrate that the D4WM isotope analysis method is an important alternative method if not one of the most effective methods for safe, cost effective, simple and unambiguous biotracer studies, without the use of undesirable radioactive isotopes. The PI will also study new laser methods for measuring circular dichroism in the gas-phase samples using thermal or polarization-grating based D4WM setups. Successful detection and analysis of optically active chiral biomedical substances at trace-concentration levels promise many applications in various biomedical areas.